CONICET | Buscador de Institutos y Recursos Humanos

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The photoreceptor that mediates this effect is the protein LOV-HK. This protein contains an N-terminal photosensory LOV domain (Light, Oxygen and Voltage), a PAS domain and a C-terminal histidine kinase domain. The molecular mechanism of the light sensing in bacteria is unknown. LOV domains consist of an ?Ñ/?Ò core where FMN is bound and a C-terminal ?Ñ-helix, called J-helix, which is involved in the transduction of the signal to the output domain of the molecule. Light absorption causes formation of a covalent bond between a conserved Cys residue and the C(4a) atom from the FMN ring. While the covalent bond formation is fast (in £gs) in all LOV proteins characterized, the recovery to the dark state varies among different LOV-containing proteins, ranging from 20-30 seconds in phototropins to hours in some prokaryotic photoreceptors. In Brucella LOV-HK the adduct state is extremely stable (does not decay significantly even for days). However, absorption spectra and 1D 1H NMR studies show that a Brucella LOV domain fragment comprising only the ?Ñ/?Ò?ncore completes the photocycle after several hours. The addition of only 20 residues corresponding to the J-helix to this core abrogates the dark recovery, showing the same truncated photocycle as the full-length LOV-HK protein. These findings indicate that the J-helix is not just a transducer of the signal to the HK domain but also an element of the sensor domain that determines its photochemical behavior. The crystal structure of the Brucella LOV domain shows the canonical fold previously observed for other LOV domains, comprising a five-stranded antiparallel £]-sheet and four ?Ñ-helices. The molecule was trapped in the dark state. There are two molecules within the asymmetric unit. The surface of contact may indicate the LOV core form a dimer. The residues that interact with FMN are conserved. The N-terminal part of the J-helix, included in the crystal structure, extends apart from the core and into the solvent. NMR spectroscopy shows that Brucella LOV core is a dimer in solution with the same folding observed in crystals, including the N-terminal fragment of the J-helix. Dynamics relaxation measurements show that the domain is rigid under dark conditions, however conformational instability was detected in the interface between monomers, shown in the crystal structure. HSQC experiments in light and dark conditions show that there are not big conformational changes upon illumination, but several residues suffer changes in their chemical shift. Most of them are contacting FMN and others are not, among which there must be the key elements in the signal transduction to the effector domain. The degree of change in the chemical shift awaits the assignment of the lit state.Light affects the virulence of Brucella abortus, the etiologic agent of brucellosis. The photoreceptor that mediates this effect is the protein LOV-HK. This protein contains an N-terminal photosensory LOV domain (Light, Oxygen and Voltage), a PAS domain and a C-terminal histidine kinase domain. The molecular mechanism of the light sensing in bacteria is unknown. LOV domains consist of an ?Ñ/?Ò core where FMN is bound and a C-terminal ?Ñ-helix, called J-helix, which is involved in the transduction of the signal to the output domain of the molecule. Light absorption causes formation of a covalent bond between a conserved Cys residue and the C(4a) atom from the FMN ring. While the covalent bond formation is fast (in £gs) in all LOV proteins characterized, the recovery to the dark state varies among different LOV-containing proteins, ranging from 20-30 seconds in phototropins to hours in some prokaryotic photoreceptors. In Brucella LOV-HK the adduct state is extremely stable (does not decay significantly even for days). However, absorption spectra and 1D 1H NMR studies show that a Brucella LOV domain fragment comprising only the ?Ñ/?Ò?ncore completes the photocycle after several hours. The addition of only 20 residues corresponding to the J-helix to this core abrogates the dark recovery, showing the same truncated photocycle as the full-length LOV-HK protein. These findings indicate that the J-helix is not just a transducer of the signal to the HK domain but also an element of the sensor domain that determines its photochemical behavior. The crystal structure of the Brucella LOV domain shows the canonical fold previously observed for other LOV domains, comprising a five-stranded antiparallel £]-sheet and four ?Ñ-helices. The molecule was trapped in the dark state. There are two molecules within the asymmetric unit. The surface of contact may indicate the LOV core form a dimer. The residues that interact with FMN are conserved. The N-terminal part of the J-helix, included in the crystal structure, extends apart from the core and into the solvent. NMR spectroscopy shows that Brucella LOV core is a dimer in solution with the same folding observed in crystals, including the N-terminal fragment of the J-helix. Dynamics relaxation measurements show that the domain is rigid under dark conditions, however conformational instability was detected in the interface between monomers, shown in the crystal structure. HSQC experiments in light and dark conditions show that there are not big conformational changes upon illumination, but several residues suffer changes in their chemical shift. Most of them are contacting FMN and others are not, among which there must be the key elements in the signal transduction to the effector domain. The degree of change in the chemical shift awaits the assignment of the lit state.Light affects the virulence of Brucella abortus, the etiologic agent of brucellosis. The photoreceptor that mediates this effect is the protein LOV-HK. This protein contains an N-terminal photosensory LOV domain (Light, Oxygen and Voltage), a PAS domain and a C-terminal histidine kinase domain. The molecular mechanism of the light sensing in bacteria is unknown. LOV domains consist of an / core where FMN is bound and a C-terminal -helix, called J-helix, which is involved in the transduction of the signal to the output domain of the molecule. Light absorption causes formation of a covalent bond between a conserved Cys residue and the C(4a) atom from the FMN ring. While the covalent bond formation is fast (in μs) in all LOV proteins characterized, the recovery to the dark state varies among different LOV-containing proteins, ranging from 20-30 seconds in phototropins to hours in some prokaryotic photoreceptors. In Brucella LOV-HK the adduct state is extremely stable (does not decay significantly even for days). However, absorption spectra and 1D 1H NMR studies show that a Brucella LOV domain fragment comprising only the /core completes the photocycle after several hours. The addition of only 20 residues corresponding to the J-helix to this core abrogates the dark recovery, showing the same truncated photocycle as the full-length LOV-HK protein. These findings indicate that the J-helix is not just a transducer of the signal to the HK domain but also an element of the sensor domain that determines its photochemical behavior. The crystal structure of the Brucella LOV domain shows the canonical fold previously observed for other LOV domains, comprising a five-stranded antiparallel β-sheet and four -helices. The molecule was trapped in the dark state. There are two molecules within the asymmetric unit. The surface of contact may indicate the LOV core form a dimer. The residues that interact with FMN are conserved. The N-terminal part of the J-helix, included in the crystal structure, extends apart from the core and into the solvent. NMR spectroscopy shows that Brucella LOV core is a dimer in solution with the same folding observed in crystals, including the N-terminal fragment of the J-helix. Dynamics relaxation measurements show that the domain is rigid under dark conditions, however conformational instability was detected in the interface between monomers, shown in the crystal structure. HSQC experiments in light and dark conditions show that there are not big conformational changes upon illumination, but several residues suffer changes in their chemical shift. Most of them are contacting FMN and others are not, among which there must be the key elements in the signal transduction to the effector domain. The degree of change in the chemical shift awaits the assignment of the lit state.